Methods of using conjugates of saccharides and acetamidino or guanidino compounds for treating bacterial infections

ABSTRACT

A method of treating a bacterial infection in an individual is provided. The method is effected by administering to the individual a therapeutically effective amount of a pharamaceutical composition including an acetamidino- or guanidino- conjugated saccharide.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to a method of treating bacterialinfections using conjugates of saccharides and acetamidino or guanidinocompounds.

Antibiotic resistance is a growing problem encountered with all classesof antibiotics. One of the first groups of antibiotics to encounter thechallenge of resistance was the aminoglycoside-aminocyclitol family.Aminoglycosides constitute a large group of biologically activebacterial secondary metabolites, which are used in the treatment ofserious bacterial infections, such as tuberculosis and nosocomialinfections.

Initially, resistance was restricted to bacterial modification of theantibiotic targets. For instance, all streptomycin-resistant M.tuberculosis strains carry point mutations leading to alterations in theribosome, the site targeted by the antibiotic agent. As newaminoglycosides came into use, chemical modification mechanisms ofresistance became more widespread. Unlike penicillin resistance whereantibiotic hydrolysis is the mechanism of action, resistance toaminoglycosides is mediated by enzymes, which catalyze co-factordependent modification of the hydroxy or amino groups of aminocyclitolresidues.

Aminoglycoside-modifying enzymes are characterized by several levels ofaminoglycoside inactivation: ATP-dependent O-phosphorylation byphosphotransferases (APH), ATP-dependent O-adenylation bynucleotidyltransferases (ANT) and acetyl CoA-dependent N-acetylation byacetyltransferases. Over 50 different enzymes found in mostGram-negative and Gram-positive bacterial pathogens have been identifiedas aminoglycoside modifiers [Shaw, K J. et al. (1993) Microbiol. Rev.57:138-163], including a chimeric enzyme, which protects strains thatcarry it from almost all available aminoglycosides.

Thus, with growing bacterial resistance to antibiotics, the challenge atpresent is to generate highly potent antibacterial agents, which areeffective at treating resistant strains and yet not toxic for use inhumans.

Several approaches have been undertaken to uncover novel antibioticagents or make presently employed antibiotic agents effective intreating resistant strains.

Aminoglycoside-Derivatives

Several aminoglycoside derivatives were designed and tested. Theeffectiveness of such novel aminoglycoside-derivatives is examined interms of antibacterial potency, degree of resistance to inactivation bymicrobial enzymes and potential toxicity. An assessment of a number ofcompounds structurally related to gentamycin, sisomicin, fortimicin andkanamycin, revealed that none had overall properties superior to theirparental compounds. In no case did a compound prove to be less toxic,and in many instances, the antibacterial potency of the newer agents waslower than that exhibited by the older aminoglycosides, while only aslight increase in resistance to inactivating enzymes was seen (reviewedin Price, K E. et al. (1986) Am. J. Med. 80:182-189).

Protein Kinase Inhibitors

Recent crystal structures of APHs, showed high similarity between APH(3′)-IIIa and protein kinases, which encouraged the use of proteinkinase inhibitors as APH inhibitors [Daigle, D M. Et al. (1997) J. Biol.Chem. 272:24755-24758]. Indeed, various inhibitors of serine/threonineand tyrosine kinases (e.g., the isoquinoline sulfonamides and theflavanoids genistein and quercetin) showed mid μM-inhibition of the APHenzymes, however reversal of antibiotic resistance was not observed.

Aminoglycoside Modifications

Synthesis of aminoglycoside molecules which have antibiotic propertiesand are poor substrates for modifying enzymes has also been attempted.For example, tobramycin and dibekacin lack the 3′-hydroxyl group whichis the site of APH(3′)-catalyzed phosphorylation of kanamycin class ofaminoglycosides, and as such are competitive inhibitors of APH(3′) andpotentially useful as antibiotic agents [McKay, G A. et al (1995) J.Biol. Chem. 270:24686-24692, Umezawa, S. et al. (1971) J. Antibiot.24:274-275]. Unfortunately, tobramycin and dibekacin serve as substratesfor other aminoglycoside kinases such as APH(2″), which are frequentlyfound in Gram-positive organisms [Daigle, D M. et at. (1999) J. Biol.Chem. 6:99-110].

In another approach, several analogues of kanamycin and neamine lackingeither the NH₂ group or the OH group in positions that are common sitesfor AAC modification, but remote to typical kinase targets hydrolysis,were synthesized [Roestamadji, J. et al. (1995) J. Am. Chem. Soc.117:11060-11069]. Several of these compounds were very poor substratesfor APH(3′)-Ia and APH(3′)-IIa, and exhibited antimicrobial activity inE. coli containing these enzymes. Although this approach is promising itis limited by the fact that most of these compounds were effectivelyphosphorylated by APH(3′)-IIIa.

While reducing the present invention to practice the present inventorshave uncovered that compositions that include an acetamidino- orguanidino-conjugated saccharide are capable of relieving and curingbacterial infections.

Thus, the present invention provides novel antimicrobial agents andmethods of using same for treating bacterial infections even when suchinfections are caused by previously resistant strains of bacteria.

SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided amethod of treating a bacterial infection in an individual, the methodcomprising administering to the individual a therapeutically effectiveamount of a pharmaceutical composition including an acetamidino- orguanidino-conjugated saccharide.

According to another aspect of the present invention there is providedan article of manufacture comprising packaging material and apharmaceutical composition identified for treatment of a bacterialinfection being contained within the packaging material, thepharmaceutical composition including, as an active ingredient, anacetamidino- or guanidino-conjugated saccharide and a pharmaceuticallyacceptable carrier.

According to further features in preferred embodiments of the inventiondescribed below the acetamidino- or guanidino-conjugated saccharide isof a formula:

According to still further features in the described preferredembodiments A is CH₃ or NH₂; X is a linear or branched C₁-C₈ alkylchain; n is an integer equal to or greater than 1; and Sac is theresidue of a mono- or oligo-saccharide.

According to still further features in the described preferredembodiments n is an integer from 1 to 6.

According to still further features in the described preferredembodiments the alkyl chain includes a side group selected from thegroup consisting of a hydroxy group, an amino group and an oxo group.

According to still further features in the described preferredembodiments the acetamidino- or guanidino-conjugated saccharide isacetamidino-conjugated saccharide and whereas A is CH₃.

According to still further features in the described preferredembodiments the Sac is a monosaccharide.

According to still further features in the described preferredembodiments the active ingredient is methyl6-deoxy-6-(N-acetamidino)-α-D-mannopyranoside.

According to still further features in the described preferredembodiments the Sac is an oligosaccharide.

According to still further features in the described preferredembodiments the oligosaccharide is a residue of an aminoglycosideantibiotic.

According to still further features in the described preferredembodiments the aminoglycoside antibiotic is selected from the groupconsisting of neomycin, kanamycin, sisomycin, fortimycin, paromomycin,neamine and gentamycin.

According to still further features in the described preferredembodiments the active ingredient is γ-(N-acetamidino) butyricacid-neomycin B.

According to still further features in the described preferredembodiments the active ingredient is tetra-γ-(N-acetamidino) butyricacid-kanamycin A.

According to still further features in the described preferredembodiments the active ingredient is guanidino-conjugated saccharide andwhereas A is NH₂.

According to still further features in the described preferredembodiments the Sac is a monosaccharide.

According to still further features in the described preferredembodiments the acetamidino- or guanidino-conjugated saccharide ismethyl 6-deoxy-6-guanidino-α-D-mannopyranoside.

According to still further features in the described preferredembodiments the active ingredient is methyl6-deoxy-6-(N-L-argininamido)-α-D-mannopyranoside.

According to still further features in the described preferredembodiments the Sac is an oligosaccharide.

According to still further features in the described preferredembodiments the oligosaccharide is a residue of an aminoglycosideantibiotic.

According to still further features in the described preferredembodiments the aminoglycoside antibiotic is selected from the groupconsisting of neomycin, kanamycin, sisomycin, fortimycin, paromomycin,neamine and gentamycin.

According to still further features in the described preferredembodiments the acetamidino- or guanidino-conjugated saccharide istetraargininamido-kanamycin A conjugate of a formula:

According to still further features in the described preferredembodiments the acetamidino- or guanidino-conjugated saccharide istriargininamido-gentamycin C conjugate of a formula:

According to still further features in the described preferredembodiments the acetamidino- or guanidino-conjugated saccharide istetraargininamido-gentamycin C conjugate of a formula:

According to still further features in the described preferredembodiments the acetamidino- or guanidino- conjugated saccharide ishexa-argininamido-neomycin B conjugate of a formula:

According to still further features in the described preferredembodiments the acetamidino- or guanidino-conjugated saccharide istetraargininamido-neamine 1 conjugate of a formula:

According to still further features in the described preferredembodiments the acetamidino- or guanidino-conjugated saccharide is apentaargininamido-paramomycin conjugate of a formula:

According to still further features in the described preferredembodiments the acetamidino- or guanidino-conjugated saccharide isγ-(N-guanidino)butyric acid-neomycin B conjugate of a formula:

According to still further features in the described preferredembodiments the acetamidino- or guanidino-conjugated saccharide is atetra-γ-(N-guanidino) butyric acid-kanamycin A conjugate of a formula:

The present invention successfully addresses the shortcomings of thepresently known configurations by providing a novel approach fortreating bacterial infections using conjugates of saccharides andacetamidino or guanidino compounds.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIG. 1 schematically illustrates the aminoglycoside-arginine conjugatesutilized by the methods of the present invention.

FIG. 2 is a sequence alignment of a portion of the RNA-binding domain ofRNase P retrieved from a number of bacterial strains. Grey boxesindicate an arginine-rich consensus sequence.

FIG. 3 is an autoradiogram depicting ptRNA processing mediated by RNaseP of various bacterial strains, in the absence and presence of indicatedconcentrations of aminoglycoside-arginine conjugates.

FIGS. 4 a-b illustrate ptRNA cleavage efficiency of E. coli RNase P as afunction of increasing concentrations [nM] of NeoR (FIG. 4 a) and R3G(FIG. 4 b).

FIG. 5 is an autoradiogram depicting the effect of variousconcentrations of NeoR and R3G on ptRNA processing mediated by humanRNase P.

FIG. 6 is an autoradiogram depicting the effect of indicatedconcentrations of polyA on the inhibition of E. coli RNase P activity byNeoR and R3G.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention can be used for the treatment of bacterialinfections. Specifically, the present invention employs conjugates ofsaccharides and acetamidino or guanidino compounds for the treatment ofvarious bacterial diseases.

The principles and operation of the present invention may be betterunderstood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details of construction and the arrangement of the components setforth in the following description or illustrated in the drawingsdescribed in the Examples section. The invention is capable of otherembodiments or of being practiced or carried out in various ways. Also,it is to be understood that the phraseology and terminology employedherein is for the purpose of description and should not be regarded aslimiting.

The aminoglycoside antibiotics are broad-spectrum antibacterialcompounds that were used extensively for the treatment of many bacterialinfections. However their increased use has led to the appearance ofresistant bacterial strains. This, together with high cytotoxicity,limited the broad clinical use of such antibiotics.

While reducing the present invention to practice, the present inventorshave uncovered that conjugates of saccharides and acetamidino orguanidino compounds, specifically, derivatives of aminoglycosides, arehighly efficient as bacteriocidal/bacteriostatic agents.

As is further detailed hereinbelow, these conjugates enable treatment ofbacterial infections even in cases where such infections are resistantto conventional antibiotic agents, or when toxicity of conventionalantibiotics prevents utilization of an aggressive treatment regimen.

Although the complete mechanism of action of these conjugates is notthoroughly understood, it is conceivable that they interfere withbacterial targets i.e., RNA-protein complexes (RNP), thus blockingvarious biological processes necessary for pathogen growth andproliferation [for further details see the background of the Examplessection and Eubank et al. (2002) FEBS Lett. 511:107-112].

A structural study of aminoglycoside-arginine conjugates (AACs) and HIVRNA target i.e., the trans-activator responsive element (TAR), enabledcharacterization of the structural determinants ofaminoglycoside-arginine conjugates which are important for substraterecognition and affinity [Litovchick A. et al. (2001) Biochemistry,40:15612-15623].

This study suggested that AAC binding is different than that of theparental aminoglycoside compounds. Binding of aminoglycoside-arginineconjugates to RNA targets is predicted to be a combination of specificbinding of one arginine moiety with the bulge of TAR-RNA andnon-specific interactions between the rest of the conjugate and the loopsegment of TAR-RNA.

Thus, specific parameters that may contribute to binding affinity of theaminoglycoside conjugates are: (i) length and rigidity of the linkerbetween the aminoglycoside core and the guanidine group of the argininemoiety; (ii) interaction between the α-amino of theaminoglycoside-arginine conjugate and the RNA target, as experimentallypredicted by structural models of NeoR binding to TAR-RNA [Litovchick A.et al. (2000) Biochemistry 39:2838-2852]; (iii) multiple contact pointsgained from the interaction of at least one arginine and the bulge ofTAR-RNA [Seewlad MJ. et al. (1998) J. Biomol. Struct. Dynamics16:683-692 and Litovchick A. et al. (2000) Biochemistry 39:2838-2852].

Thus, according to one aspect of the present invention, there isprovided a method of treating a bacterial infection in an individual.Preferred individual subjects according to the present invention aremammals such as canines, felines, ovines, porcines, equines, bovines,humans and the like.

The term “treating” refers to alleviating or diminishing a symptomassociated with a bacterial infection. Preferably, treating cures, e.g.,substantially eliminates, the symptoms associated with the infectionand/or substantially decreases bacterial load in the infected tissue.

Bacterial infections treated according to the present invention includeopportunistic aerobic gram-negative bacilli such as the generaPseudomonas, bacterial infection caused by P. aeruginosa, bacterialinfections caused by gram-positive bacilli such as that of the genusMycobacterium, and mycobacteria, which causes tuberculosis-likediseases. A variety of bacterial infections may be treated by the methodof the present invention, these include: M. tuberculosis, M. leprae, M.Intracellulare, M. smegmatis, M. bovis, M. kansasii, M. avium, M.scrofulcium, or M. africanum.

The method includes administering to the individual a therapeuticallyeffective amount of an acetamidino- or guanidino-conjugated saccharide.

The saccharide according to the present invention may be a simplemonosaccharide such as (i) pentose, e.g., arabinose, xylose, ribose andthe like; (ii) disaccharide such as hexose, e.g., sucrose, maltose,lactose, cellobiose and the like; (iii) trisaccharide, e.g.,mannotriose, raffmose, meleziose and the like; or (iv) atetrasaccharide, e.g., amylopectin, Syalyl Lewis X (SiaLex) and thelike. Alternatively, the saccharide can be a saccharide derivative suchas, but not limited to, glucosides, ethers, esters, acids and aminosaccharides.

A preferred saccharide of the present invention is a naturalaminoglycoside antibiotic such as, but not limited, kanamycin, neomycin,seldomycin, tobramycin, kasugamycin, fortimicin, gentamycin,paromomycin, neamine and sisomicin. Alternatively, semi-syntheticderivatives of aminoglycosides such as amikacin, netilmicin and the likecan also be used.

The saccharide residue may be linked to a spacer (X) through anysuitable group, for example through an alkylene chain or, preferably,through an acylamino group.

The aminoglycoside-arginine conjugates (AACs) of the present arepreferably of the following general formula:

wherein A is NH₂ and X is (CH₂)₃—CH(NH₂)—C(═O)—.

Several conjugation schemes can be employed, including conjugation ofone or more arginine derivative moieties to one or more saccharidecores. The conjugates preferably include short chains of L and D (n=1-6)arginines, although longer chains of n=10 or even n=20 are alsoenvisaged. Alternative, conjugates can be α,ω-diamino acids of varyinglength such as β-alanine, ornithine and lysine (2,3 and 4 methylenegroups, resepectively) or γ-amino acids such as glycine (aminoaceticacid), β-amino propionic acid or γ-amino butyric acid conjugated toaminoglycosides converted at the terminal amino groups into guanidine orN-acetamidino moieties. Examples of conjugates which can be utilized bythe present invention include but are not limited to:6-deoxy-6-(N-acetamidino)-α-D-mannopyranoside, γ-(N-acetamidino) butyricacid-neomycin B, tetra-γ-(N-acetamidino) butyric acid-kanamycin A,6-deoxy-6-guanidino-α-D-mannopyranoside,6-deoxy-6-(N-L-argininamido)-α-D-mannopyranoside,monoarginineamido-kanamycin A, monoarginineamido-gentamycin C,monoarginineamido-neomycin B, monoarginineamido-paramomycin,diarginineamido-kanamycin A, diarginineamido-gentamycin C,diarginineamido-neomycin B, diarginineamido-paramomycin,tetraargininamido-kanamycin A, triargininamido-gentamycin C,tetraargininamido-gentamycin C, hexa-argininamido-neomycin B,tetraargininamido-neamine 1, pentaargininamido-paramomycin,γ-(N-guanidino) butyric acid-neomycin B, tetra-γ-(N-guanidino) butyricacid-kanamycin A and the like [International Pat. NO: WO 00/39139,Litovchick et al. (1999) FEBS Lett. 445:73-79, Litovchick et al. (2000)Biochemistry 39:2838-2852 and Lapidot A. and Litovchick A. (2000) DrugDevelop. Res. 50:502-515, Cabrera C. et al. (2000) AIDS Res. Hum.Retroviruses 16:627-634, Litovchick et al. (2001) Biochemistry40:15612-15623, Cerebra et al. (2002) Antiviral research 53:1-8;Carriere et al. (2002) RNA 8:1267-1279 and Catani et al. (2002) J.Neurochemistry in-press].

The active ingredient (AAC) of the method of the present invention canbe administered to an individual per se, or as part of a pharmaceuticalcomposition where it is mixed with a pharmaceutically acceptablecarrier.

As used herein a “pharmaceutical composition” refers to a composition ofone or more of the active ingredients described hereinabove, orphysiologically acceptable salts or prodrugs thereof, with otherchemical components such as physiologically suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of a compound to an organism.

Hereinafter, the phrases “pharmaceutically acceptable carrier” and“physiologically acceptable carrier” are used interchangeably to referto a carrier or a diluent that does not cause significant irritation toa treated individual and does not abrogate the biological activity andproperties of the active ingredient.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration ofactive ingredients. Examples, without limitation, of excipients includecalcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils and polyethyleneglycols.

Techniques for formulation and administration of the pharmaceuticalcompositions of the present invention may be found in “Remington'sPharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latestedition, which is incorporated herein by reference.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, intestinal or parenteral delivery, includingintramuscular, subcutaneous and intramedullary injections as well asintrathecal, direct intraventricular, intravenous, inrtaperitoneal,intranasal, or intraocular injections.

Alternately, one may administer a pharmaceutical composition in a localrather than systemic manner, for example, via injection of thecomposition directly into the area of infection often in a depot or slowrelease formulation, such as described below.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredient intocompositions which, can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the invention may be formulatedin aqueous solutions, preferably in physiologically compatible bufferssuch as Hank's solution, Ringer's solution, or physiological salinebuffer. For transmucosal administration, penetrants appropriate to thebarrier to be permeated are used in the formulation. Such penetrants aregenerally known in the art.

For oral administration, the pharmaceutical composition can beformulated by combining the active agents with pharmaceuticallyacceptable carriers well known in the art. Such carriers enable thepharmaceutical composition used by the method of the invention to beformulated as tablets, pills, dragees, capsules, liquids, gels, syrups,slurries, suspensions, and the like, for oral ingestion by a patient.Pharmacological compositions for oral use can be made using a solidexcipient, optionally grinding the resulting mixture, and processing themixture of granules, after adding suitable auxiliaries if desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose compositions such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarbomethylcellulose; and/or physiologically acceptable polymers such aspolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acidor a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active ingredient doses.

Pharmaceutical compositions, which can be used orally, include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, theactive ingredients may be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the agents for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from a pressurized pack or a nebulizer with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. Inthe case of a pressurized aerosol, the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, e.g., gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the active ingredient and a suitable powderbase such as lactose or starch.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.

The compositions described herein may be formulated for parenteraladministration, e.g., by bolus injection or continuous infusion.Formulations for injection may be presented in unit dosage form, e.g.,in ampoules or in multidose containers with optionally, an addedpreservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain fornulatoryagents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active ingredient in water-soluble form.Additionally, suspensions of the active ingredient may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidsesters such as ethyl oleate, triglycerides or liposomes. Aqueousinjection suspensions may contain substances, which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol or dextran. Optionally, the suspension may also containsuitable stabilizers or formulations, which increase the solubility ofthe active ingredient to allow for the composition of highlyconcentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-,freewater, before use.

The composition of the present invention may also be formulated inrectal compositions such as suppositories or retention enemas, using,e.g., conventional suppository bases such as cocoa butter or otherglycerides.

In addition to the formulations described previously, a composition ofthe present invention may also be formulated for local administration,such as a depot composition. Such long acting formulations may beadministered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecomposition may be formulated with suitable polymeric or hydrophobicmaterials (for example, as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives such as sparinglysoluble salts. Formulations for topical administration may include, butare not limited to, lotions, suspensions, ointments gels, creams, drops,liquids, sprays emulsions and powders.

The pharmaceutical compositions herein described may also comprisesuitable solid of gel phase carriers or excipients. Examples of suchcarriers or excipients include, but are not limited to, calciumcarbonate, calcium phosphate, various sugars, starches, cellulosederivatives, gelatin and polymers such as polyethylene glycols.

Pharmaceutical compositions suitable for use in context of the presentinvention include compositions wherein the active ingredients arecontained in an amount effective to achieve the intended purpose. Morespecifically, a therapeutically effective amount means an amount ofactive ingredient effective to prevent, alleviate or ameliorate symptomsof disease or prolong the survival of the subject being treated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed examples provided herein (see Example 1 of the Examplessection).

For any composition used by the methods of the invention,. thetherapeutically effective amount or dose can be estimated initially fromcell culture assays and cell-free assays (See Example 2 and Example 3 ofthe Examples section). For example, a dose can be formulated in animalmodels to achieve a circulating concentration range that includes theIC₅₀ as determined in in-vitro assays. Such information can be used tomore accurately determine useful doses in humans.

The AACs utilized by the present invention exhibit far greater affinitytowards their cellular targets than their parental compositions (seeExample 1 of the Examples section below), and as such, lowconcentrations/quantities thereof may be used in treatment of variousbacterial infections, thereby avoiding cytotoxicity. In particular,cytotoxicity analysis showed that NeoR is not toxic to mice whenadministered as two single doses of 25 mg/kg body weight for theduration of two hours [Litovchick A. et al. (2001) Biochemistry40:15612-15623].

Regardless, toxicity and therapeutic efficacy of the pharmaceuticalcompositions described herein can be determined by standardpharmaceutical procedures in experimental animals, e.g., by determiningthe IC₅₀ and the LD₅₀ (lethal dose causing death in 50% of the testedanimals) for a subject ingredient. The data obtained from assays can beused in formulating a range of dosage for use in human. The dosage mayvary depending upon the dosage form employed and the route ofadministration utilized. The exact formulation, route of administrationand dosage can be chosen by the individual physician in view of thepatient's condition. (See e.g., Fingl, et al., 1975, in “ThePharmacological Basis of Therapeutics”, Ch. 1 p.1).

Dosage amount and interval may be adjusted individually to provideplasma levels of the active ingredient, which are sufficient to maintainthe 21 required effects, termed the minimal effective concentration(MEC). The MEC will vary for each composition, but can be estimated fromin vitro data; e.g., the concentration necessary to achieve 50-90%inhibition (see Example 1 of the Examples section). Dosages necessary toachieve the MEC will depend on individual characteristics and route ofadministration. HPLC assays or bioassays can be used to determine plasmaconcentrations.

Dosage intervals can also be determined using the MEC value.Compositions should be administered using a regimen, which maintainsplasma levels above the MEC for 10-90% of the time, preferable between30-90% and most preferably 50-90%.

It is noted that, in the case of local administration or selectiveuptake, the effective local concentration of the drug may not be relatedto plasma concentration. In such cases, other procedures known in theart can be employed to determine the effective local concentration.

Depending on the severity and responsiveness of the infection to betreated, dosing can also be a single administration of a slow releasecomposition, with course of treatment lasting from several days toseveral weeks or until cure is effected or diminution of the infectionstate is achieved.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the infection,the manner of administration, the judgment of the prescribing physician,etc.

Compositions of the present invention can be packaged in a dispenserdevice, as one or more unit dosage forms as part of an FDA approved kit,which preferably includes instruction for use, dosages and counterindications. The kit can include, for example, metal or plastic foil,such as a blister pack suitable for containing pills or tablets, or adispenser device suitable for use as an inhaler. The kit may also beaccompanied by a notice associated with the container in a formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals, which notice is reflective of approval by theagency of the form of the compositions or human or veterinaryadministration. Such notice, for example, may be of labeling approved bythe U.S. Food and Drug Administration for prescription drugs or of anapproved product insert. Compositions comprising an active ingredientsuitable for use with the present invention may also be prepared, placedin an appropriate container, and labeled for treatment of an indicateddisease or condition.

Many diseases and conditions associated with bacterial infections aredifficult if not impossible to treat using commercially availableantibiotics due to bacterial resistance and drug-associatedcytotoxicity.

The bacteriocidal activity of acetoamido- or guanido-saccharideconjugates makes such compounds highly suitable for treating bacterialinfections even in cases where prolonged treatment regimens arenecessary. As such, these compounds may play a pivotal role in thefields of therapy and antibiotic design in years to come. Furthermore,the incomparable affinity and specificity that the conjugates of thepresent invention have towards bacterial RNA (see Example 2 of theExamples section) may serve as a basis for the development of adiagnostic assay for premature detection of bacterial infections. Theproposed novel assay may be far more specific and reliable than presentmethods.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Maryland (1989); Perbal, “A Practical Guideto Molecular Cloning”, John Wiley & Sons, New York (1988); Watson etal., “Recombinant DNA”, Scientific American Books, New York; Birren etal. (eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes 1-111 Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishelland Shiigi (eds), “Selected Methods in Cellular Immunology”, W. H.Freeman and Co., New York (1980); available immunoassays are extensivelydescribed in the patent and scientific literature, see, for example,U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987;3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345;4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader. Allthe information contained therein is incorporated herein by reference.

BACKGROUND

RNase P is a ubiquitously expressed enzyme, which catalyzes processingof the 5′ termini of precursor tRNAs (ptRNAs) and other cellular RNAs(e.g., p4.5S RNA) which are involved in protein biosynthesis [xiao, S.et al. (2001) J. Cell Physiol. 187:11-21, Altman, S. (1999) “The RNAWorld” Cold Spring Harbor Laboratory Press, Cold Sping Harbor , N.Y.2^(nd) edition 351-380 and Harris, M E. Et al. (1998) “RNA Structure andFunction” Cold Spring Harbor Laboratory Press, Cold Spring Harbor , N.Y.309-337]. The bacterial RNase P holoenzyme is composed of a catalyticRNA moiety (˜350-400 nucleotides) and a protein co-factor (˜110-150amino acid residues).

RNase P recognizes the ptRNA structure via interactions between thecatalytic RNA subunit and the T- and acceptor-stems mainly, althoughresidues in the 5′-leader sequence as well as the 3′-terminal sequencealso contribute to such interactions. The protein subunit of RNase Papparently also affects substrate recognition as well as the range ofsubstrates, which can be used by RNase P. Although the RNA subunit cancatalyze the ptRNA processing reaction in-vitro under non-physiologicalconditions [Guerrier-Takada, C. et al. (1983) Cell 35:849-857], probablydue to its role in substrate recognition, the protein subunit is vitalfor RNase P activity in-vivo [Schedl, P. et al. (1973) Proc. Natl. Acad.Sci. 70:2091-2095 and Kurz, J C. et al. (2000) Curr. Opin. Chem. Biol.4:553-558].

Thus, inhibition of bacterial RNase P activity is a major goal for drugdesigners due to its essential role in bacterial protein synthesis. Inaddition, due to its unique structure, which is not shared with thehuman enzyme, the bacterial holoenzyme, represents an excellent drugtarget.

EXAMPLE 1 Inhibition of In-Vitro Reconstituted Bacterial RNase PActivity by Aminoglycoside-Aginine Conjugates

The ability of Aminoglycosides-arginine conjugates (AACs) to inhibitbacterial RNase P was investigated due to observations that (i)aminoglycosides interact with the RNA subunit of E. coli RNase P invitro and interfere with its ptRNA-processing activity [Mikkelsen, N E.et al. (1999) Proc. Natl. Acad. Sci. 96:6155-6160] and (ii) sequenceanalysis of the protein subunit of RNase P from various bacterialspecies revealed an arginine-rich consensus, encompassed in theRNA-binding domain (RNR motif) of the RNase P protein co-factor [seeFIG. 2, Vioque, A. et al. (1988) J. Mol. Biol. 202:835-848 and Gopalan,V. (1997) J. Mol. Biol. 267:818-829].

Materials and Methods

Reagents

Oligonucleotides for PCR were synthesized at HHMI Biopolymer/KeckFoundation Resource laboratory, Yale university School of medicine, NewHaven Conn. Restriction and modifying enzymes were obtained from NewEngland Biolabs, Beverlt Mass. and Gibco Life Technologies, Rockville,Md. T7 RNA ploymerase and Rnasin were purchased from Promega, Madison,Wis. Hi Trap columns and γ-[³²P]-GTP were obtained from AmershamPharmacia Biotech. All other reagents used were purchased fromSigma-Aldrich St. Louis, Mo. and Fisher Biotech, Pittsburgh, Pa.

RNA, Protein and Inhibitor Preparation Synthesis and Purification

Polynucleotide sequences of Neisseria gonnorhoeae, Porphyromonasgingivalis and Streptococcus pneumoniae (SEQ ID NOS: 1, 3 and 5,respectively) expressing amino acid (SEQ ID NOS: 2, 4 and 6,respectively) subunits of RNase P were PCR amplified using standard PCRmethodology. The genes encoding the RNA subunit of RNase P were clonedinto pUC19 under the transcriptional control of a T7 RNA polymerasepromoter. T7 RNA polymerase-mediated run-off in vitro transcription wasperformed on individual clones to generate the respective RNase P RNAs,which were then purified using Quick Spin columns. cDNAs encoding theprotein subunits of the various bacterial species were subcloned intoeither expression vectors: pCRT7TOPO or pBAD (Invitrogen, Carlsbad,Calif.). Proteins were over-expressed in E. coli as His₆-tagged fusionproteins and purified to homogeneity using a combination of cationexchange and immobilized metal affinity chromatography.

DNA sequences were confirmed by DNA sequencing and molecular weight ofthe respective proteins was determined by electrospray ionization massspectrometry.

RNase P from E. coli was prepared and purified according to Vioque, A.et al. (1988) J. Mol. Biol. 202:835-848 and Gopalan, V. (1997) J. Mol.Biol. 267:818-829.

Synthesis of NeoR and R3G was described elsewhere [Litovchick, A. et al.(1999) FEBS Lett. 445:73-79, Litovchick, A. et al. (2000) Biochemistry39:2838-2852, Lapidot, A. et al. (2000) Drug Develop. Res. 50:502-515and Litovchick, A. et al. Biochemistry in press].

ptRNA^(Tyr)su3+ was prepared by in vitro transcription of FokI-digestedpUC19TyrT [Vioque, A. et al. (1988) J. Mol. Biol. 202:835-848].

RNase P Activity Assay

RNase P activity was determined in the presence or absence of AACinhibitors suspended in 50 mM Tris-Hcl (7.2), 5 % (w/v) polyethyleneglycol 8000, 1 mM NH₄Cl, 10 mM spermidine, 10 mM MgCl₂. Reactions werecarried under multiple-tumover conditions (for example, 100 nM ofradio-labeled ptRNA^(Tyr)su3+ and 0.5 nM E. coli RNase P holoenzyme).

Following holoenzyme assembly, AAC inhibitors were added to the reactionmixture and incubated for 5 minutes prior to the addition of[³²P]-ptRNA^(Tyr)su3+ substrate. Reactions were allowed to proceed forthe indicated times and were terminated by adding a quenching dye [7 MUrea, 10 mM EDTA, 10% (v/v) phenol]. Reaction products were resolved bygel electrophoresis (8% polyacrylamide/7 M Urea) and auto-radiogramswere obtained.

Extent of substrate cleavage was quantified using a PhosphorImager(Molecular Dynamics) and ImageQuant softwares. Initial cleavage velocitywas calculated only from those reactions exhibiting substrate cleavagelower than 30%.

Results

Reconstituted RNase P activity was tested in the presence or absence ofindicated concentrations of AAC inhibitors. As shown in FIG. 3, in theabsence of AAC inhibitors, radiolabeled-ptRNA^(Tyr)su3+ was wellprocessed by E. coli RNase P and in particular by enzymes derived fromN. gonnorhoeae and S. pneumoniae; less effective was ptRNA processingmediated by P. gingivalis. Addition of AAC inhibitors, either 500 nMNeoR or 1500 nM R3G to the reaction mixture resulted in nearly completeinhibition of RNase P processing activity; RNase P activity derived fromP. gingivalis was less susceptible to the addition of the indicatedinhibitors.

IC₅₀ values (i.e., concentration of inhibitor required to reduceenzymatic activity by 50% as observed in the absence of inhibitor) ofNeoR and R3G were determined in the presence of increasingconcentrations of either inhibitors. Initial reaction velocities weredetermined at various concentrations of each inhibitor. As shown in FIG.4 a-b, NeoR (FIG. 4 a) and R3G (FIG. 4 b) inhibited E. coli RNase Pactivity with IC₅₀ values of 125 nM and 300 nM, respectively. Furtherresults suggest that IC₅₀ values for NeoR and R3G-mediated inhibition ofvarious bacterial RNase P are in the sub-micromolar concentration range(FIG. 4). The IC₅₀ value of NeoR is 100-fold lower than that presentedby the parental aminoglycoside [FIG. 4, Mikkelsen, Nebr. et al. (1999)Proc. Natl. Acad. Sci. 96:6155-6160].

EXAMPLE 2 Specificity of Aminoglycoside-Arginine Conjugates TowardsProkaryotic RNase P

RNase P functions as an RNP complex in all living organisms, howeverconsiderable variation in composition and structure exist. Compared tothe simple composition and structure of bacterial RNase P (e.g., one RNAsubunit: one protein subunit), the human holoenzyme is characterized bya higher level of complexity [Xiao, S. et al. (2001) J. Cell Physiol.187:11-21]. In addition to a 340-nucleotide long RNA subunit, at leasteight protein subunits ranging in size from 14 kDa to 115 kDa are foundin association with the RNA subunit of human RNase P. Interestingly,none of the protein subunits posses the conserved arginine-rich tractfound in bacterial RNase P. Moreover, the eukaryotic RNA subunit ofRNase P is catalytically inactive in-vitro unlike its bacterialcounterpart.

In order to determine if the AAC inhibitors of the present inventioncross-react with human RNase P, the activity of a partially purifiedhuman enzyme was tested in the absence or presence of variousconcentrations of NeoR and R3G.

Results are shown in FIG. 5. Although human RNase P activity was largelyunaffected at concentrations, which were 10-fold greater than the IC₅₀values of NeoR and R3G for E. coli RNase P, a nearly complete inhibitionof the human enzyme was observed at NeoR and R3G concentrations of 7.5μM.

From these results it can be construed that the AACs utilized by thepresent invention are more effective in inhibiting bacterial RNase Pthan human RNase P.

EXAMPLE 3 Specificity of Aminoglycoside-Arginine Conjugates TowardsRNase P

Positively charged compounds may serve as general inhibitors of anynegatively charged biological molecule and as such of RNA. In order todetermine whether the aminoglycoside-arginine conjugates of the presentinvention are specific inhibitors of RNase P, the inhibitory effect ofNeoR and R3G on E. coli RNase P was examined in the presence or absenceof various concentrations of positively charged molecules.

As shown in FIG. 6,addition of an 18-mer polyA oligonucleotide (lanes24) or L-Arginine (lanes 10-11) did not inhibit RNase P specificactivity even at 10-fold excess concentration over that of the ptRNAsubstrate used in the assay. Furthermore, addition of as much as 1 μMpoly A RNA, failed to alter the ability of NeoR or R3G to inhibit E.coli RNase P (lanes 5-10).

These results are consistent with the finding that the inhibitorypotential of NeoR and R3G vary dependent on the source of enzyme (see,FIG. 3 and FIG. 5), and with the reported observation that a 10-foldexcess of tRNA had no effect on the ability of R3G to disrupt the RNPcomplex formed between HIV TAR RNA and Tat-derived peptide [LitovchickA. (2001) Biochemistry submitted for publication], again indicating thataminoglycoside-arginine conjugates have only a weak affinity to tRNAs.

Therefore it may be concluded that the inhibition of bacterial RNase Pby NeoR and R3G is not due to their ability to bind non-specifically theptRNA substrate and thereby interfere with RNase P catalysis.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents, patent applicationsand sequences identified by their accession numbers mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent, patent application or sequence identified by theiraccession number was specifically and individually indicated to beincorporated herein by reference. In addition, citation oridentification of any reference in this application shall not beconstrued as an admission that such reference is available as prior artto the present invention.

1. A method of treating a bacterial infection in an individual, themethod comprising administering to the individual a therapeuticallyeffective amount of a pharamaceutical composition including, as anactive ingredient, an acetamidino- or guanidino-conjugated saccharide.2. The method of claim 1, wherein said acetamidino- orguanidino-conjugated saccharide is of a formula:

wherein A is CH₃ or NH₂; X is a linear or branched C₁-C₈ alkyl chain; nis an integer equal to, or greater than 1; and Sac is the residue of amono- or oligo-saccharide.
 3. The method of claim 2, wherein n is aninteger from 1 to
 6. 4. The method of claim 2, wherein said alkyl chainincludes a side group selected from the group consisting of a hydroxygroup, an amino group and an oxo group.
 5. The method of claim 2,wherein said acetamidino- or guanidino-conjugated saccharide isacetamidino-conjugated saccharide and whereas A is CH₃
 6. The method ofclaim 5, wherein Sac is a monosaccharide.
 7. The method of claim 6,wherein said acetamidino- or guanidino-conjugated saccharide is methyl6-deoxy-6-(N-acetamidino)-α-D-mannopyranoside.
 8. The method of claim 5,wherein Sac is an oligosaccharide.
 9. The method of claim 8, whereinsaid oligosaccharide is a residue of an aminoglycoside antibiotic. 10.The method of claim 9, wherein said aminoglycoside antibiotic isselected from the group consisting of neomycin, kanamycin, sisomycin,fortimycin, paromomycin, neamine and gentamycin.
 11. The method of claim10, wherein said acetamidino- or guanidino-conjugated saccharide isγ-(N-acetamidino) butyric acid-neomycin B.
 12. The method of claim 10,wherein said acetamidino- or guanidino-conjugated saccharide istetra-γ-(N-acetamidino) butyric acid-kanamycin A.
 13. The method ofclaim 2, wherein said acetamidino- or guanidino-conjugated saccharide isguanidino-conjugated saccharide and whereas A is NH₂.
 14. The method ofclaim 13, wherein Sac is a monosaccharide.
 15. The method of claim 14,wherein said acetamidino- or guanidino-conjugated saccharide is methyl6-deoxy-6-guanidino-α-D-mannopyranoside.
 16. The method of claim 14,wherein said acetamidino- or guanidino-conjugated saccharide is methyl6-deoxy-6-(N-L-argininamido)-α-D-mannopyranoside.
 17. The method ofclaim 14, wherein Sac is an oligosaccharide.
 18. The method of claim 17,wherein said oligosaccharide is a residue of an aminoglycosideantibiotic.
 19. The method of claim 18, wherein said aminoglycosideantibiotic is selected from the group consisting of neomycin, kanamycin,sisomycin, fortimycin, paromomycin, neamine and gentamycin.
 20. Themethod of claim 19, wherein said acetamidino- or guanidino-conjugatedsaccharide is tetraargininamido-kanamycin A conjugate of a formula:


21. The method of claim 19, wherein said acetamidino- orguanidino-conjugated saccharide is triargininamido-gentamycin Cconjugate of a formula:


22. The method of claim 19, wherein said acetamidino- orguanidino-conguated saccharide is tetraargininamido-gentamycin Cconjugate of a formula:


23. The method of 19, wherein said acetamidino- or guanidino-conguatedsaccharide is hexa-argininamido-neomycin B conjugate of a formula:


24. The method of claim 19, wherein said acetamidino- orguanidino-conjugated saccharide is tetraargininamido-neamine 1 conjugateof a formula:


25. The method of claim 19, wherein said acetamidino- orguanidino-conjugated saccharide is pentaargininamido-paramomycinconjugate of a formula:


26. The method of claim 19, wherein said acetamidino- orguanidino-conjugated saccharide is γ-(N-guanidino) butyric acid-neomycinB conjugate of a formula:


27. The method of claim 19, wherein said acetamidino- orguanidino-conjugated saccharide is tetra-γ-(N-guanidino) butyricacid-kanamycin A conjugate of a formula:


28. An article of manufacture comprising packaging material and apharmaceutical composition identified for treatment of a bacterialinfection being contained within said packaging material, saidpharmaceutical composition including, as an active ingredient, anacetamidino- or guanidino-conjugated saccharide and a pharmaceuticallyacceptable carrier.
 29. The article of manufacture of claim 28, whereinsaid acetamidino- or guanidino-conjugated saccharide is of a formula:

wherein A is CH₃ or NH₂; X is a linear or branched C₁-C₈ alkyl chain; nis an integer equal to, or greater than 1; and Sac is the residue of amono- or oligo-saccharide.
 30. The method of claim 29, wherein n is aninteger from 1 to
 6. 31. The article of manufacture of manufacture ofclaim 29, wherein said alkyl chain includes a side group selected fromthe group consisting of a hydroxy group, an amino group and an oxogroup.
 32. The article of manufacture of claim 29, wherein acetamidino-or guanidino-conjugated saccharide ingredient is acetamidino-conjugatedsaccharide and whereas A is CH₃.
 33. The article of manufacture of claim32, wherein Sac is a monosaccharide.
 34. The article of manufacture ofclaim 32, wherein said acetamidino- or guanidino-conjugated saccharideis methyl 6-deoxy-6-(N-acetamidino)-α-D-mannopyranoside.
 35. The articleof manufacture of claim 32, wherein Sac is an oligosaccharide.
 36. Thearticle of manufacture of claim 35, wherein said oligosaccharide is aresidue of an aminoglycoside antibiotic.
 37. The article of manufactureof claim 36, wherein said aminoglycoside antibiotic is selected from thegroup consisting of neomycin, kanamycin, sisomycin, fortimycin,paromomycin, neamine and gentamycin.
 38. The article of manufacture ofclaim 37, wherein said acetamidino- or guanidino-conjugated saccharideis γ-(N-acetamidino) butyric acid-neomycin B.
 39. The article ofmanufacture of claim 37, wherein said acetamidino- orguanidino-conjugated saccharide is tetra-γ-(N-acetamidino) butyricacid-kanamycin A.
 40. The article of manufacture of claim 29, whereinsaid acetamidino- or guanidino-conjugated saccharide isguanidino-conjugated saccharide and whereas A is NH₂.
 41. The article ofmanufacture of claim 40, wherein Sac is a monosaccharide.
 42. Thearticle of manufacture of claim 41, wherein said acetamidino- orguanidino-conjugated saccharide is methyl6-deoxy-6-guanidino-α-D-mannopyranoside.
 43. The article of manufactureof claim 41, wherein said acetamidino- or guanidino-conjugatedsaccharide is methyl 6-deoxy-6-(N-L-argininamido)-α-D-mannopyranoside.44. The article of manufacture of claim 30, wherein Sac is anoligosaccharide.
 45. The article of manufacture of claim 44, whereinsaid oligosaccharide is a residue of an aminoglycoside antibiotic. 46.The article of manufacture of claim 45, wherein said aminoglycosideantibiotic is selected from the group consisting of neomycin, kanamycin,sisomycin, fortimycin, paromomycin, neamine and gentamycin.
 47. Thearticle of manufacture of claim 46, wherein said acetamidino- orguanidino-conjugated saccharide is tetraargininamido-kanamycin Aconjugate of a formula:


48. The article of manufacture of claim 46, wherein said acetamidino- orguanidino-conjugated saccharide is triargininamido-gentamycin Cconjugate of a formula:


49. The article of manufacture of claim 46, wherein said acetamidino- orguanidino-conjugated saccharide is tetraargininamido-gentamycin Cconjugate of a formula:


50. The article of manufacture of claim 46, wherein said acetamidino- orguanidino-conjugated saccharide is hexa-argininamido-neomycin Bconjugate of a formula:


51. The method of claim 46, wherein said acetamidino- orguanidino-conjugated saccharide is tetraargininamido-neamine 1 conjugateof a formula:


52. The method of claim 46, wherein said acetamidino- orguanidino-conjugated saccharide is pentaargininamido-paramomycinconjugate of a formula:


53. The article of manufacture of claim 46, wherein said acetamidino- orguanidino-conjugated saccharide is γ-(N-guanidino) butyric acid-neomycinB conjugate of a formula:


54. The article of manufacture of claim 46, wherein said acetamidino- orguanidino-conjugated saccharide is tetra-γ-(N-guanidino) butyricacid-kanamycin A conjugate of a formula: